Clustering of tRNA genes in Paracentrotus lividus mitochondrial DNA

CurrentGenetics

Curr Genet (1988) 13 : 91-96

© Springer-Verlag 1988

Clustering of tRNA genes in Paracentrotus lividus mitochondrial D NA P. Cantatore x , M. Roberti, G. Rainaldi, C. Saccone, and M. N. Gadaleta 1 Dipaxtimento di Biochimica e Biologla Molecolare, Universit/t di Bari, Via Amendola 165/A, 1-70126 Bari, Italy Centro di Studio sui Mitocondri e Metabolismo Energetico, 1-70126 Bari, Italy

Summary. We have determined the base sequence of the restriction fragment Barn1-2 (3,593) of Paracentrotus lividus (sea urchin) mtDNA. This fragment contains, in addition to genes previously identified (part of the 12S rRNA, ND1 and part of the ND2 mRNA), a cluster of 15 tRNA genes located between the 12S and ND1 genes. Also to be found in the tRNA gene cluster, between the tRNA TM and tRNA Pr° genes, is a sequence of 134 bp which constitutes the only non-coding region of this DNA so far identified. The distinctive organization of the tRNA genes and the extreme size reduction of the non-coding region suggest the existence o f unique mechanisms for the regulation of gene expression in this organism.

tion than found in mammals and the ÷D~rALeu tI~z-xCUN, tRNA Glu, tRNA TM and tRNA pr° genes have not been found in positions corresponding to their mammalian counterparts. In order to complete the P. lividus mtDNA gene order we focused our attention on a fragment, Baml-2 which was the only one whose size did not account for the genes identified in it (part of the 12S rRNA, ND1 and the 5' portion of the ND2 mRNA). Analysis of the Barn1-2 nucleotide sequence has allowed us to establish the complete gene organization of the P. lividus mt genome: this fragment contains, in addition to genes previously identified, a cluster of 15 tRNA genes and an intergenic region of 134 bp which does not seem to have a structural role.

Key words: mtDNA - Sea urchin - Gene organization - tRNA gene clustering

Materials and methods

Introduction Recent work in our laboratory based on a combination of RNA-DNA hybridization and DNA sequencing has provided information on the location of most of the RNA genes in the mitochondrial (mt) genome of the sea urchin P. lividus (Cantatore et al. 1987a). These genes are arranged in a fashion particular to this organism. The two rRNA genes - 16S and 12S - are not adjacent as in the majority of animal mtDNAs sequenced to date, but are separated by a region of about 3,000 base pairs (bp) containing the genes for two subunits of the NADH-dehydrogenase complex (ND1 and ND2). Moreover, the ND4L gene does not flank ND4, but is located between the tRNA Arg and COIl genes. Other differences concern the tRNA genes: the tRNA Arg gene is in a different posi-

Restriction enzymes and DNA modifying enzymes were from Boehringer or New England Biolabs; ~-32pdNTP from Amersham. The other chemicals were of the purest grade available. Digestion with restriction enzymes, separation, elution and cloning of DNA fragments were performed as described by Maniatis et al. (1982). To ob~in the base sequence of Baml-2, the fragment was digested for varying periods of time at 30 °C with 1 U/pmol of Bal31 nuclease. The digestion products were cloned in the plasmid pUC8 and end-sequenced by using the Sanger procedure (1977) adapted for double-stranded templates (Chen and Seeburg 1985). Sequence analysis was carried out on a VAX/ VMS 4.2 operating system by using the programs GLORIA and ACNUC recently developed in our laboratory (Gouy et al. 1985). Results and discussion

a) Gene organization of the P. lividus mtDNA Baml-2 fragment 1". lividus mtDNA was cut with BamHI, PstI and EcoRI,

Offprint requests to: P. Cantatore

generating seven restriction fragments that were cloned

•

1~S ,RNA

~

[ ' ~ t~ ' *~T'~G IkC TAAA[A T I k G/n0&A21 Cnr;&kAAA I ('1~~t(~AYCT A0CAAAAAAA I I'~AI~C~k I+~n (I fi(,AAA/+ I ACl0t 0CCAflCCACP,P~1~4~ I I A I A(;[; 1 ACt T (,C~AkA0~ 1 AA/+TAAkA(:O~ T r IO o 30 4O 50 ~0 70 1o 90 100 11o 12o

ktkaA00OC~I~/+OkAAAAI*n/+IIIG/+~IOISGCT~C*n~T~GIG T/+/+A~AAC ~kTCT ~ G ~ k r , A ~ C A I / + A ~ T C A ~ A ~ I

130

I¢0

t~O

160

I20

190

190

~?TTi;A~ CCUCn~kAACTA/+0~CCIA~k~C 210 2~0 ~30 2~0

20~

*~A1t/+~kT6C¢¢1Ac1ATAcTT~0Ac~T~/+/+cA*c~1AA~C/+C~AnAn~A(+~A;:AAkP,~AA~11T~A/+Acrc~*~O~1~n~m;T~¥T~cA1k~:cTc~crn~Anknc1T~cc~T

250

~40

2?0

2~0

290

3OO

310

320

33O

34o

390

36O

I o A * r c o19! ~+11c cat" 60 k A CAP.I;TC/+r,CAAP,1111 G? I l k * TC/+fl¢ T T r, T~ ~A I:C A 15C T r;tl T AAGTr;T AC I T C 11 A~fl A* A a t 1001: Tt:P,T AUnnAA 40 T TCCCk O/+CCI CAt+4 T CA I+0 3~0 3110 1190 400 410 4~0 430 440 4~0 460 470 490

o I Gc A&c Ac A I AAOC TGOGnAI n/a0 Tnan(: I A P.AAI C T T t T ~/+&CI;AOP,P.~(;I:t~AAAA T k(; AAA 1 GAAACA& T T CI T I A I;⁡T I U(;A 11 [;k~C FI0 TA Al'; CCCCAAA 1 A~&Ak~ 1 A 01]GC • 9O ~00 ~|0 5~0 130 ~40 510 ~0 S?O ~eo 190 600 t RNAGlU._..~ 10Ak*AnAOC TCTnGAA! OCQI ACACAVCflC£Cr+TCACTCTCOCC TAr+IT1 TAACTTAAG1AkG fig nan AAA&0 T C0 TAAI:SC AAT AGIII:kC~CGnAAGCCT0 Ok &kA Pil;,CT CC TATAn i [ 610

62O

~30

640

650

&60

470

~10

690

700

710

7~0

tRNAthr~

~,i

T AC&AC/+k0&GC T I T TCA4~r+CTCIAI+P,T T T P,A0 T ?AA/+ATC 1C~AI I~I;A A I;C 1~ AI: ++l~p.l:GT T0&AanC T CAAI k n l 1 A UAI]I~GCCA/+r, TC 110T&Ak(: ? t C~,~¢,k OA r,c,0 T T A~k 230

740

?SO

200

770

710

710

I00

110

920

930

9~'0"

~ - c c c 1 c t ¢ * . o o c +]t r t o TAr T TCCC~ t C~¢;A*C/+TOTCC c i t e TCTCTC~'~I CUI C(;G TC(~GnI CCO0 GC6 T TTCTI;101 ¥/+l:l+'T1 T1 T ! CT TAT6 OGOGOOO0060 n o 0 a n o n ; n o n o o n 160 170 leo 199 900 910 920 930 940 950 960

IRNAPro~ 0ATI~/+¥~TTTATAIA1/+/+(~ACACnITAI~AAAOAAIAnlTTAAnAAAA/+ AAC~ TTTOOP+AGTTflTAOAC IA/+(;II;AAAA c i ACTICTTIOP/NTTAAGO&t~IhP+0A&IIOAAC 970 910 990 1000 I010 1020 ' " 1030 IO40 1050 1060 1070 1010 ~tRNA

GIn

tRNAA*n~

~k ¢ AmC* +~G/+AOTCA/+AflT 1 CO TCOTT 11 r Or,AT +AAACt/+C TP,C YTelGeA(;AAAt~Tt:0~+ T0 TA0C l AAn I r11AmAC,(iC1,CTTGP+t:(;fiTTAAt~CAA0Ok s I &~,Thi+nO T~ 6A~/+CC T 1000 It00 I110 IL20 1130 1140 IISO 1160 1170 I110 1190 1200

I R N A L e U t C~~1 A I C 11CC CA ( ~ p ? t;A&A ~A GCA AA 0 T I~fl I T &A I fi CA~&AgAC I r *OdA 1 T T 11 CA T CPsA~Gn 11C A AC TCCT T CT T T C& P.CItIGTAmT T CC? &/+a/+AT T T~&C T T ~GI;k I T T CTGC7 T GC& 1210

1720

1230

1240

1250

1260

~tRNA All t RNATrp~ /+AO~&&ACa~;'~T~CACTAA&CTAaAI'ICI:AqA/+G/+I1 T1/+AUTT/+&ATAA&C 1330

1340

1390

1360

1270

121J0--"

1290

1300

1310

13~0

tRNACY*~ /+AAaC TTC&~/+O~TTTTTA AAGA/+TOII/+~/+CTTCTTAOCTCTTI~GGCTTT01&OTGTAAAI~A~

1370

1380

"

1390

1400

1410

1420

~430

1440

.~___tRNA v,I A T 1 T 10 I;,/+11GC &A& r Cc T r A(;/+ triP+All I TAOTTCA Tr OCC/+A/+nC~r~:~An,+,:4Accco/+AI r OC/+C~/+nTOr n Ace TC 0 0 1 0 T/+~/+k0 m00A/+C.TTACT&&CTk OC t AT01r: TTam+AT 1450

1460

1470

14B0

1490

1500

IS10

1520

1530

1540

1590

1960

tRNAt-Met.__.~ A 1 A TaT A TA 1~AGCAGA01 AAnC t A An TOt; I PlAOCT T T T ItOflC I CAT ACCCCA/+I;/+A10aA/+O.~ 1/+II/+A/+CCTCCC T CT OC T Ti~/90AAOCc ir)c TnGAk I T T&ACCItJCAA I T T T ~14~1~CTill 1970

1500

1590

1600

1610

1620

1630

1640

16,50

1660

1670

1610

. t _ _ _ t RNA'I+~ t RHATYr_.__~ ACAAC~TA~AGTT~ TA TTTTA/+CTM~ACCTC 1p'CTTTCCTTTTilk/+ TAAI~PlTIinCT0/+n/+FIAACAA~iCGOTOnATTnTAAATCCAIACAC++A/+OQTTI~A/+TCCCTY TTCT 11~TT+qiTCTT~ 1690 1700 1710 1220 1730 1740 1750 1760 1720 1700 1790 1109 1R N A G I ~ . _ _ . ~ l R N A L e " I U~. ~) I~ TC I ATI;AGI&CAACAOTACCT T TO/+CT1CCA/+1 C/++t/+T/+flC T CT I AacI~&AAA YCT mOnA T/+a~la r RiTt~C T 4/+AA T/+OCAAPl0TGOI T I~AT OCAa/+AGGCCTAAG/+CCT TCCTATC/+/+ 1110 1920 1130 1140 1150 1960 11170 1180 1100 1900 1910 1920 NOI II r Y L • T V L I~ L L ~ F L V P V L L 0 V /+ F L ~" L V R K V L &GOT T(~AACTCC[ I T 1 1 1 T A ~ 1 ~ A 1 ~ A C ~ T A T T 1 ~ T T ~ A ~ A n T A C T ~ G A A ~ T A ~ T T T C T T T T T T ~ T i ~ C C A G T 1 ~ T T C T T ~ C A ~ T ~ T T 1 ~ CC/+CTCTAGTTa&AC6A/+AGOT&TT 1930

1940

1950

1960

1970

1991

1990

2000

2010

2020

2030

2040

G ¥ m 0 F R K G • I¢ | V 0 • Y G L L 0 p ~" /+ D G I+ K L F l K F. T L K P T N 0 • 60 Gr TACk T GCA/+I+TCCI3A&A(I(tOACC/+~AkA T/+OI y elIAC CCIIIT Iln¢1CTCTT/+CAACCC T TT GCAOACGOCCTT/+~I3CT TT I+TAT T/+/+60AA/+CT T TAAAOCCCT CCACAOCATC&CC

Y L • ~ L S P L L F M /+ L /+ L L L ¥ M L H • U H T P T L N F 2 L S L L V L 0 L A T A C I+TGT T T Y T T T T0 TC TCCCC1 II I TA T T TA i"GGCACT T OC~TT G0TCT TOTG&AA/+T T0 4 TGCCT GI A CACA(;/+P,C/+/+CCT T/+/+ATT I CCAACTGTC1CTG0 TAC T ~0 TC0T TGG~CT 2120 2110 2190 2200 2211 2220 2230 2240 2250 22&0 2271 2210

S S I. S V ¥ & 1 L II S G U /+ & H S K Y II L I P, 4 1 It k I/ +t I1 T I 1 Y £ II L & L 0 l CA++AP,r I ATCCS t r t A t G C C * t A C l ++nOA; ¢ I nG/+T000CCOCC~A*¥C TA/+GT/+C~¢6 T T/+/+T/+n~Ancn/+ T/+CoAoCCn t/+OCCCAA*CT/+T T TCCT/+CGAOATTAOCCTCOC&TT 2290 2300 2310 2320 2330 2,140 23,';0 2360 2270 2310 2390 2400 I

t

t

s

t

I

1

!

T

S

S

~

m

t

Y

Y

I

m

M

/+

0

t

F

9

u

F

A

t

9

c

L

•

t

r

y

M

~

V

S

A/+ I C T T 4C T T TC&£ T T/+r T A TAA I r~cc#n/+AG6 T T T AAC T TAAC# TA T/+I AA TflAACCCCCAAGAGTT T T CCT O01 T T GC/+CT */+GATe+nOT TC£AC 1GT T 1 1 A I * T T T OAT 1TGI CT C 2410 ~420 2430 ~44t ~4~0 2460 2470 2410 2490 2~00 2510 2520 T L A [ 1 N R A P F ~ L T • 0 [ | [ 1 V s n Y N P ~ y k ~1 G P F k A~rccl~oc~nAa*c*~A~c~Ir, ctecet I ~1~rA/+~A~ma~A~knTcr~T~n~ArccnGArA~+~c~AArAT~cAn~An~+mC~:~TT~nC~T~r~ 2930 ?~40 ~S~0 ~60 ?~70 2"J80 2~90 2+00 2/910 2620 A

N

I

1

L

m

M

L

•

S

P

V

I

F

L

0

P+ P

M

P

A

14

~"

L

F

P

L

9

V

L

T

I

0

I

F"

1 k I~ y I TkTTr.'C.TI~AAi+A 26]0

L

K

T

2640 L

I

Q

l GC I A kAA T kA T t I [ A k T A mATOT(; T T T I CALl I +aT CA I T T T T C T I nOTOnCeCA Ttl/+CClIOC/+AAC04AC r A r I r c c c c t m/++k/+oT T T T AAe T A r T G0 k T TOA/+O/+CCACT C T T T T 00 T 2650 2660 267O 2610 2690 2700 2710 2720 2730 2740 22~0 2760 L r I U V 9 * A Y I" ~ r n Y n e t ~ F L T U K S ~ t P L S I e A t C A 1 T I T I r 11 ~A~ t CCTAr G/nOr * C 0 0 n ¢ t ¢ , C C T A r c c c r . 0 / + T I TCOGT*Tn/+TCAACTAA~TT TC TA&CTT1;/+A04;/101;TACC r hCC/+C1CT~r,/+r TGO*OCCCTATOCOCC~CAA TA/+CTAT 2~0 ~10 2790 ~000 ~910 ~20 ~130 2~40 2~S0 2~60 29~0 2980

.

.

.

.

.

.

.

.

.

.

.

.

RNA ~-.--i~

ND2H " ~ ,

Arl TACr OACI:C TAGGA+~r TTA #C T YCCCC r/+ TT TI A~I~AOC Tr tic TCCO+tl/+AOr: TTBOOTATCT/niler o k TPIAr 1/+0/+c TAT~ ~pA(;tll~ I T/+A~TT~ ~ T TI ~ + ~ [ [ TIAr o c ACCA&AT 2990 2900 ~910 2920 2930 ~940 2950 21~0 2970 2990 2W0 3000 S | ~" i • I F v V S 0 r I I v v II /+ • M g F V 1 M L 0 L [ L 9 T L A L P l L GA 1 A TC TII TC +T T T 1 0 T T T T T GACAG TCA 1 C TC TGGAACT/+T/A/+TAg 1 el;IT 11 CAI;CAOAA/+&,++TGf i t TC 01 T/+TATI;I;C TGGGACT/+0~I~+C7 AAI;mACCCT00CC ~+TAATCCCAk Ti~CT 3010 30-+0 3O20 3040 3050 3060 3070 3080 3090 3100 31 tO 3120 C F T • R M I ~ /+ T I K V F L V o /+ F i l A /+ L L L N II m L I 0 /+ V F 0 +; w r 1 +in t r c t o c T I CA t a COp,CO++~An^ rAO/+AOC/+~+CC/+TAA*O r ~ c Tr c o TTn r TO/+AGet r r r AOAOCCOC t C TOCt/+ T TAAA/+AOtO COCTA/At~CA * o c t I n k t t r t CG+ r,A t c T r + 3130 3140 ltSO 1160 3170 3111O 3190 3200 3210 3220 3230 3~4